Figure 5.

Complementation with ASAP1 [K75E, K76E, K79E] does not rescue the effects of ASAP1 downregulation on actin stress fibers. U2OS cells were stably transduced with tet-inducible empty vector, full-length WT (HA-ASAP1), or HA-ASAP1 [K75E, K76E, K79E] lentiviruses and transfected with control (diCtrl) DICER substrate RNA duplex or diRNA against the 3′UTR region of human ASAP1 (diASAP1). After 24 h, expression of the empty vector or ASAP1 was induced or not with doxycycline (100 ng/ml) for 48 h. The cells were subsequently processed for immunoblotting or immunofluorescence experiments. A, immunoblot summarizing the efficiency of knockdown of endogenous ASAP1 and doxycycline-induced overexpression of empty vector or ASAP1 and mutants. Membranes were probed with rabbit anti-ASAP1 antibody to detect endogenous and exogenous ASAP1, mouse anti-HA antibody to detect expression of transgenes, and anti-mouse GAPDH as a loading control. B, cells were plated on fibronectin in serum-free media, fixed and stained with fluorescent phalloidin for F-actin. After 5.5 h in serum-free media, U2OS develop a robust stress fiber network (diCtrl), which is greatly diminished in diASAP1-treated cells. Open arrowheads indicate the loss or reduction of stress fibers, whereas closed arrowheads indicate rescue of stress fibers. The graphs summarize the quantification of the number of actin filaments per cell in each treatment group from individual experiments. The scale bar represents 10 μm. N = 3 independent experiments for empty vector control and five independent experiments for WT and mutant ASAP1, with 10 to 30 cells for each condition. See also Fig. S1. Statistics are based on One-way ANOVA with Dunnet’s multiple comparison test n.s. – not significant, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Dox, doxycycline; F-actin, filamentous actin.